Alpha-glucosidase inhibitors from fungal hydroxylation of tibolone and hydroxytibolones

ABSTRACT

Sixteen new and one known metabolites were obtained by incubation of tibolone and hydroxytibolones with various fungi. Their structures were elucidated by means of a homo and heteronuclear 2D NMR and by HREI-MS techniques. The relative stereochemistry was deduced by 2D NOESY experiment. These metabolites have shown promising inhibitory activities against α-glucosidase enzyme. Seven metabolites exhibited potent inhibitory activity against the α-glucosidase enzyme and were thus found useful in the treatment of diabetes mellitus and other diseases related to the activity of α-glucosidase enzyme.

SUMMARY OF INVENTION

Microbial transformation is an effective tool to synthesize many steroidal drugs with potential biological activities. Such studies are primarily useful in the generation of hydroxylated metabolites for drug toxicity studies. Fungi, bacteria and yeast have been utilized successfully as in vitro models to mimic and predicts the metabolic fate of drugs and other xenobiotics in mammalian systems. Previously, many biotransformational studies on various 17α-ethynyl steroids had been carried out with various fungal and bacterial strains, which afforded hydroxylation at various positions.

Tibolone (1) is a synthetic steroid that combines estrogenic and progestogenic properties with androgenic property, which mimic the action of a male sex hormone. The in vivo metabolism of tibolone (1) in human had been studied with the reference to its three metabolites, 3α-hydroxytibolone, 3β-hydroxytibolone and Δ⁴-tibolone.

In the current study, tibolone (1) was used as a structural probe to identify its metabolites produced through microbial model, and to further investigate the difference between microbial transformation and human metabolism. These metabolic studies showed novel hydroxylation at various positions (Compounds 4-20), in which only Compound 4 was reported previously as metabolite in human metabolism. Compound 1 when incubated with Rhizopus stolonifer, Fusarium lini, Cunninghamella elegans and Gibberella fujikuroi, resulted in the formation of a library of hydroxyl derivatives. These hydroxytibolones have also targeted for inhibition of α-glucosidase enzyme.

Glucosidase enzymes are involved in several biological processes such as the intestinal digestion, the biosynthesis of glycoproteins and the lysosomal catabolism of the glycoconjugates (Homonojirimycin isomers and N-alkylated homonojirimycins: structural and conformational basis of inhibition of glycosidases. Asano N, Nishida M, Kato A, Kizu H, Matsui K, Shimada Y, Itoh T, Baba M, Watson A A, Nash R J, Lilley P M, Watkin D J, Fleet G W., J Med. Chem. 1998 Jul. 2; 41(14):2565-71). Intestinal α-glucosidases are involved in the final step of the carbohydrate digestion to convert these into monosaccharides which are absorbed from the intestine.

As a result of the catalysis produced by α-glucosidase enzyme in the final step in the digestive process of carbohydrates, its inhibitors can retard the uptake of dietary carbohydrates and suppress postprandial hyperglycemia, and could be useful to treat diabetic and/or obese patients [Novel α-glucosidase Inhibitors with a tetrachlorophthalimide Skeleton., S. Sou, S. Mayumi, H. Takahashi, R. Yamasak, S. Kadoya, M. Sodeoka, and Y. Hashimoto, Bioorg. Med. Chem. Lett., 2000, 10, 1081].

The α-glucosidase inhibitors are effective in lowering the insulin release, insulin requirement and some can lower plasma lipids. The acarbose is a very widely prescribed drug in the management of the type II diabetes and recently a U.S. Pat. No. 6,387,361 to Rosner describes the use of acarbose in the treatment of obesity. According to the criteria issued by WHO (World Health Organization) based on a glucose tolerance test, diabetes mellitus and impaired glucose tolerance (hereinafter sometimes referred to as IGT) are distinguished by the fasting blood glucose level and the blood glucose level 2 hours after glucose loading. Patients with IGT have high blood glucose levels compared to those of patients with diabetes mellitus, and are reported to be at increased risk of developing diabetes mellitus and complications of arteriosclerotic diseases. In particular, it is known that patients with IGT who have blood glucose levels of 170 mg/dl or above at 2 hours following glucose loading, i.e., patients with high-risk IGT, may develop diabetes mellitus at a high rate [Diabetes Frontier, p. 136, 1992]. With regard to voglibose which is an α-glucosidase inhibitor, there are reports of studies on effects of voglibose for insulin-resistant IGT and diabetes [Yakuri-to-Chiryo (Japanese Pharmacology & Therapeutics), 24 (5):213 (1996); Metabol. Exp. Clin., 45:731, 1996]. Voglibose (AO-128) is also known to have effects of lowering blood glucose level and improving glucose tolerance in rats [Yakuri-to-Chiryo (Japanese Pharmacology & Therapeutics), 19 (11): 161 (1991); Journal of Nutrition Science and Vitaminology, 45 (1): 33 (1992)]. On the contrary, it has also been reported that the effect of voglibose in improving glucose tolerance could not be verified in human [Rinsho-Seijinbyo, 22 (4): 109 (1992)]. An antibiotic pradimicin Q as α-glucosidase inhibitor is described in the U.S. Pat. No. 5,091,418 to Swada.

In addition, they have also been used as antiobesity drugs, fungistatic compounds, insect antifeedents, antivirals and immune modulators [Glycosidase inhibitors and their chemotherapeutic value, Part 1. el Ashry E S, Rashed N, Shobier A H., Pharmazie. 2000 April; 55(4):251-620]. The antiviral activity due to inhibition of α-glucosidase results form abnormal functionality of glycoproteins because of incomplete modification of glycans. Suppression of this process is the basis of antiviral activity [A glucosidase-inhibitors as potential broad based antiviral agents, Anand Mehta, Nicole Zitzmann, Pauline M. Rudd, Timothy M. Block, Raymond A. Dwek, Febs Letters 430 (1998) 17-22] and decrease in growth rate of tumors [Inhibition of experimental metastasis by an alpha-glucosidase inhibitor, 1,6-epi-cyclophellitol. Atsumi S, Nosaka C, Ochi Y, Iinuma H, Umezawa K. Cancer Res. 1993 Oct. 15; 53(20):4896-9]. The α-glucosidase inhibitor N-(1,3-dihydroxy-2-propyl)valiolamine is described as a promoter of calcium absorption in the U.S. Pat. No. 5,036,081.

In the present invention is reported a surprising discovery was made when it was discovered that the several metabolites of tibolone (1) or its hyroxyderivates obtained by fermentation with various fungi are potent inhibitors of alpha-glucosidase enzyme, a property of these chemicals that has never been reported before in the prior art. Table 1 lists are metabolites of tibolone and their activity against alpha glucosidase enzyme.

TABLE 1 Novel metabolites of tibolone and their activity against alpha-glucosidase enzyme Compound Name Novel Active Compound 1 Tibolone (C₂₁H₂₈O₂) No No Compound 2 3β-hydroxytibolone (C₂₁H₃₀O₂) No No Compound 3 3α-hydroxytibolone (C₂₁H₃₀O₂) No No Compound 4 Δ⁴-Tibolone (C₂₁H₂₈O₂) No Yes Compound 5 6β-Hydroxytibolone (C₂₁H₂₈O₃) Yes Yes Compound 6 15β-Hydroxytibolone (C₂₁H₂₈O₃) Yes No Compound 7 Δ^(1,4)-Tibolone (C₂₁H₂₆O₂) Yes Yes Compound 8 10β-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃) Yes No Compound 9 11α,15β-Dihydroxytibolone (C₂₁H₂₈O₄) Yes No Compound 10 11α,15β-Dihydroxy-Δ⁵-tibolone (C₂₁H₂₈O₄) Yes No Compound 11 Δ⁵-Tibolone (C₂₁H₂₈O₂) Yes No Compound 12 6β-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃) Yes Yes Compound 13 6α-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃) Yes Yes Compound 14 15α-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃) Yes Yes Compound 15 6α-Hydroxy-Δ^(1,4)-tibolone (C₂₁H₂₈O₃) Yes Yes Compound 16 6β-Methoxy-Δ⁴-tibolone (C₂₂H₃₀O₃) Yes No Compound 17 3β,6β-Dihydroxytibolone (C₂₁H₃₂O₃) Yes Not studied Compound 18 3α-Hydroxy-Δ⁵-tibolone (C₂₁H₃₂O₂) Yes Not studied Compound 19 3α,6β-Dihydroxy-Δ⁴-tibolone (C₂₁H₃₂O₃) Yes Not studied Compound 20 3α,11α-Dihydroxy-Δ⁴-tibolone (C₂₁H₃₂O₃) Yes Not studied α-Glucosidase (EC 3.2.1.20) is a typical exo-type glycosidase that releases α-glucosides from the nonreducing end side of the substrates. Diabetes mellitus is a chronic metabolic disorder characterized by high blood glucose levels. Tibolone (1) is used effectively for the treatment of menopausal symptoms and in the prevention of osteoporosis, as a hormone replacement therapy (HRT). The hormone replacement therapy (HRT) effects on glucose metabolism in non-diabetic obese postmenopausal women.

Here we are reporting a new class of α-glucosidase inhibitors i.e., 17α-ethynyl steroids along with comparison of the results with the standard inhibitors of this enzyme.

DETAILS OF INVENTION

Fermentation of tibolone (1) with Rhizopus stolonifer (TSY 0471) yielded two new mono-hydroxylated metabolites, Compounds 4 and 5, and a known metabolite, Compound 6. The HREIMS of Compound 4 exhibited the molecular ion (M⁺) at m/z 328.2171, corresponding to the formula C₂₁H₂₈O₃, which indicated that a new oxygen functionality was introduced into the molecule during fermentation period. The IR absorptions were attributed to hydroxyl (3381 cm⁻¹) and carbonyl (1705 cm⁻¹) functionalities, respectively. The ¹H NMR spectrum, compared with that of the substrate, showed a new signal of OH-bearing methine proton at δ 4.04, resonating as a doublet (J=4.0 Hz) with its corresponding carbon resonating at δ 65.9 in ¹³C NMR spectrum which was assigned to C-6 on the basis of HMBC correlations of H-6 (δ 4.04) with C-5 (δ 122.5) and C-10 (δ 128.4). In the ¹H-¹H COSY 45° spectrum, the aforementioned methine proton showed correlation with the C-7 methine proton resonated at δ_(H) 2.0. The stereochemistry of C-6 hydroxyl group was determined to be axial by the NOESY correlations between H-6 (δ 4.04) and H-19 (δ 0.76). The above spectral data concluded that Compound 4 has an —OH group at C-6 position as compared to compound 1 and was deduced to be a new metabolite.

The HREIMS of Compound 5 showed the M⁺ at m/z 328.2070, indicating an increment of 16 mass units as compared to compound 1 in accordance to formula C₂₁H₂₈O₃. The ¹H and ¹³C NMR data of 5 revealed the presence of a new OH-bearing methine group that resonated at δ_(H) 4.06 (m, W_(1/2)˜10.8 Hz) and δ_(C) 65.5 and deduced for C-15 on the basis of HMBC spectrum correlations, which showed correlation of C-16 protons (δ_(H) 2.24, 1.7) and C-14 methine proton (δ_(H) 1.85) with C-15 (δ_(C) 65.5). The stereochemistry of the newly introduced C-15 hydroxyl group was deduced as 0 on the basis of NOESY correlations between H-15 (δ_(H) 4.06) and H-14 (δ_(H) 1.85) and multiplicity of H-15 signal at δ 4.06 (W_(1/2)˜10.8 Hz). From these spectral data, the new compound 5 was deduced to 7α-methyl-17α-ethynl-15β,17β-dihydroxy-19-norandrost-5(10)-en-3-one.

The incubation of compound 1 with Fusarium lini (NRRL 68751) for 6 days also led to the isolation of a UV active Compound 6 exhibiting the M⁺ at m/z 312.2023 in HREIMS spectrum (C₂₁H₂₈O₂). The ¹H NMR spectrum showed a singlet for an olefinic proton at δ 5.82. Its broad-band decoupled ¹³C NMR spectrum showed, in comparison with that of the substrate 1, the disappearance of one quaternary carbon signal resonating at δ 128.2 for C-10 and appearance of an olefinic methine carbon at δ 126.4 which was assigned to the C-4 on the basis of HMQC spectrum, indicating the migration of the C-5/C-10 double bond to C-4/C-5. Thus creating an α,β-unsaturation in Compound 6. The axial orientation of C-10 proton was assigned on the basis of NOESY coupling between H-10 (δ 2.31) and H-8 (δ 1.64). The above spectral data supported the structure of a known Compound 6 as 7α-methyl-17α-ethynl-17β-hydroxy-19-norandrost-4-en-3-one previously isolated during human metabolism of tibolone.

The incubation of 1 (600 mg) with Cunninghamella elegans (TSY 0865) for six days yielded Compounds 7-10 (FIG. 1). The HREIMS of compound 7 showed the M⁺ at m/z 310.2004, in accordance with the formula C₂₁H₂₆O₂. Comparison between the ¹H and ¹³C NMR data of compounds 6 and 7, Compound 7 showed the presence of two additional olefinic signals at δ 7.12 (dd, J=8.4, 4.7 Hz, H-1) and 6.62 (dd, J=8.4, 2.6 Hz, H-2) in the ¹H NMR spectrum with the corresponding carbons resonated at δ 153.4 (C-1) and 127.0 (C-2), respectively, in the ¹³C NMR spectrum in compound 7. The presence of a double bond between C-1 and C-2 was further deduced from ¹H-¹H COSY 45° correlations between H-1 (δ 7.12), H-2 (δ 6.62) and H-10 (δ 2.42). While HMBC experiment showed interactions of H-1 with C-2 (δ 127.0) and C-10 (δ 43.0). The final structure of Compound 7 was deduced to be 7α-methyl-17α-ethynl-17β-hydroxy-19-norandrost-1,4-dien-3-one.

The HREIMS of Compound 8 showed the M⁺ at m/z 328.2090, in agreement with the formula C₂₁H₂₈O₃ indicating an introduction of a new oxygen in the molecule, probably in the form of a hydroxyl group. However the ¹H NMR spectrum displayed no resonance for OH-bearing methine proton, but ¹³C NMR spectrum showed a downfield oxygen-bearing quaternary carbon resonated at δ 70.3, which was assigned to C-10 through its HMBC interactions with H-1 (δ 2.36, 2.29) and H-4 (δ 5.77). The 10β-hydroxylation was deduced by the β-SCS (substituents chemical shift) of −5.1, −5.4 and −6.9 ppm for C-2, C-8 and C-11, respectively, and by the downfield shifts of C-1 and C-9 (+5.1 and +7.5, respectively) with respect to the ¹³C NMR chemical shifts in compounds 4 and 8. The spectral data supported the structure of a new Compound 8 as 7α-methyl-17α-ethynl-10β,17β-dihydroxy-19-norandrost-4-en-3-one.

The HREIMS of Compound 9 showed the M⁺ at m/z 344.2212 supporting the formula C₂₁H₂₈O₄, indicated that two oxygen had been incorporated into the molecule. The ¹H and ¹³C NMR displayed two OH-bearing methine groups resonating at δ_(H) 3.43 (ddd, J=15.1, 11.1, 5.0 Hz); and 4.10 (m, W_(1/2)˜8.82 Hz) and δ_(C) 66.1 and 65.4, respectively. The ¹H-¹H COSY 45° spectrum showed correlations of H-11 (δ 3.43) with H-9 (δ 1.62) and H₂-12 (δ 2.05, 1.51), and of H-15 (δ 4.10) with H-14 (δ 1.80) and H₂-16 (δ 2.01, 1.55). Hydroxylations at C-11 and C-15 was further supported by HMBC assignments, which has exhibited correlations of H₂-12 (δ 2.05, 1.51) and Me-18 (δ 0.90) with C-11 (δ 66.1), and correlation of H-14 (δ 1.80) with δ 65.4 (C-15). The axial orientation of C-11 proton was deduced on the basis of NOESY correlation of H-11 (δ 3.43) with Me-18 (δ 0.90) and multiplicity of H-11 signal resonating at δ_(H) 3.43 (ddd, J=15.1, 11.1, 5.0 Hz),³ while β-stereochemistry of the newly introduced OH group at C-15 was deduced by the NOESY correlations between H-14 (δ 1.80) and H-15 (δ 4.10) and multiplicity of H-15 signal resonating at δ 4.10 (W_(1/2)˜8.8 Hz). The β-orientation of C-10 proton was similar to compound 6 (FIG. 3). According to this spectral data, the structure was deduced to be 7α-methyl-17α-ethynl-11α,15β,17β-trihydroxy-19-norandrost-5(10)-en-3-one.

The HREIMS of Compound 10 showed the M⁺ at m/z 344.2341, support formula C₂₁H₂₈O₄, with an increment of 32 a.m.u. The UV spectrum showed a weak absorption at 202 nm, while IR showed absorptions at 3312 (OH), 1722 (C═O) and 1652 (C═C) cm⁻¹. The ¹H NMR spectrum showed an upfield doublet of olefinic methine proton at δ 5.42 (J=4.2 Hz, H-6), which showed COSY 45° correlations with H-7 (δ 1.83). Two additional OH-bearing methine protons resonating at δ 3.40 (ddd, J=15.3, 11.0, 4.57 Hz) and 3.91 (m, W_(1/2)˜9.9 Hz) were unambiguously assigned to H-11 and H-15 through 2D NMR and ¹³C NMR spectra. The stereochemistry of newly introduced hydroxyl group at C-11 was deduced to be a (equatorial) on the basis NOESY correlation between H-11 (δ 3.40) and H-18 (δ 0.94) and larger coupling constants (J=15.3 Hz) of H-11 signal. The β-orientation of the OH group at C-15 was deduced on the basis of NOESY correlation between H-14 (δ 1.85) and H-15 (δ 3.91) and multiplicity of H-15 signal, resonating at δ 3.91 (m, W_(1/2)˜9.9 Hz). The axial β-orientation of C-10 proton was deduced through NOESY cross peaks between H-10 (δ 2.46) and H-8 (δ 1.59). Based on the above mentioned spectral data, the structure was deduced as 7α-methyl-17α-ethynl-11α,15β,17β-trihydroxy-19-norandrost-5-en-3-one.

Tibolone (1) was fermented with Gibberella fujikuroi (ATCC 10704) for 12 days yielding six new mono-hydroxylated Compounds 11-16. The HREIMS of Compound 11 showed the M⁺ at m/z 312.1456 with corresponding formula C₂₁H₂₈O₂. The ¹H NMR spectrum showed an upfield doublet of olefinic proton at δ 5.33 (J=4.7 Hz) which was assigned to H-6 through its ¹H-¹H COSY 45° correlation with H-7 (δ 1.85). The ¹³C NMR spectrum showed low-field methine carbon resonated at δ 123.4. A double bond between C-5 and C-6 was deduced through HMBC coupling between H-6 (δ 5.33) and C-7 (δ 34.6). According to the spectral data, the structure was deduced as 7α-methyl-17α-ethynl-17β-hydroxy-19-norandrost-5-en-3-one.

Compounds 12 and 13 were found to be epimers and differentiated on the basis of ¹H NMR and NOESY experiments. The ¹H NMR spectrum of Compound 12 displayed a doublet at δ 4.05 (J=4.0 Hz), while compound 13 exhibited a doublet at δ 3.60 (J=6.4 Hz), while ¹³C NMR spectra of both the isomers 12 and 13 showed OH-bearing methine carbons resonating at δ 65.9 and 70.0, respectively. The position of the newly introduced hydroxyl at C-6 in both isomers was inferred from the HMBC coupling. The relative configuration in compound 12 of the new hydroxyl group at C-6 was inferred on the basis of coupling pattern and NOESY correlations between H-6 (δ 4.05) and C-19 methyl protons (δ 0.75), while NOESY spectrum of compound 13 also displayed correlations between H-6 (δ 3.60) and C-7 methine proton (δ 1.98). The above spectral data concluded that Compounds 12 and 13 have an —OH group at C-6 position with different orientations.

The molecular formula of Compound 14 was established as C₂₁H₂₈O₃ by HREIMS (m/z 328.2132). The ¹H and ¹³C NMR spectra of compound 14 exhibited a OH-bearing methine group resonated at δ_(H) 4.02 (m, W_(1/2) 14.9 Hz); δ_(C) 65.9 and was unambiguously assigned to C-15 on the basis of two-dimensional NMR experiments. In the ¹H-¹H COSY 45° spectrum, the aforementioned methine proton showed correlation with the C-14 methine proton resonating at δ_(H) 1.72. This was further supported by the HMBC spectrum, which exhibited correlations of C-14 proton (δ_(H) 1.72) with C-15 (δ_(C) 65.9). The α-relative configuration of OH group at C-15 was deduced on the basis of multiplicity of H-15 signal resonating at δ_(H) 4.02 (m, W_(1/2)˜14.9 Hz).¹² This spectral data led to the structure 14 as 7α-methyl-17α-ethynl-15β,17β-dihydroxy-19-norandrost-4-en-3-one.

The HREIMS of Compound 15 showed the M⁺ at m/z 326.2231 (C₂₁H₂₈O₃). Two additional doublets in the ¹H NMR spectrum for two olefinic protons appeared at δ 7.08 (dd, J=8.18, 4.2 Hz) and 6.76 (d, J=8.22 Hz) and one new hydroxymethine proton, appeared at δ 3.81 (d, J=6.4 Hz). The DEPT spectrum of compound 15 showed two olefinic methine carbon signals at δ 154.2 and 126.5, corresponding to a double bond between C-1/C-2, as deduced on the basis of HMBC correlations of H-1 (δ 7.08) with C-2 (δ 126.5) and, of H-2 (δ 6.76) with C-1 (δ 154.2) and C-4 (δ 125.7). One OH-bearing methine carbon resonated at δ 69.8 was assigned to C-6 based on ³J correlations between H-6 (δ 3.81) and C-4 (δ 125.7). The α-orientation of newly hydroxyl group at C-6 was deduced to be equatorial on the basis of NOESY correlations between H-6 (δ 3.81) and H-7 (δ 1.95). According to these spectral studies, the structure of Compound 15 was deduced to be 7α-methyl-17α-ethynl-6α,17β-dihydroxy-19-norandrost-1,4-dien-3-one.

The HREIMS of Compound 16 showed the M⁺ at m/z 343.2356 corresponding to the formula C₂₂H₃₀O₃. The ¹H NMR spectrum of Compound 16 showed the presence of a methoxy singlet resonating at δ 3.47, while geminal methoxy protons were resonated at δ 3.93 (d, J=3.9 Hz). The ¹³C NMR spectrum showed a methoxy carbon signal at δ 57.6 and a methoxy-bearing carbon resonated at δ 70.2. The position of the newly introduced methoxy group at C-6 was deduced through HMBC interactions of H-6 (δ 3.93) with C-4 (δ 126.7). The β-orientation of the newly introduced OCH₃ group at C-6 was deduced on the basis of NOESY correlations between H-6 (δ 3.93) and C-19 methyl protons (δ 0.77). The above mentioned spectral data led to conclude that Compound 16 has the structure as 7α-methyl-17α-ethynl-6β-methoxy-17β-hydroxy-19-norandrost-4-en-3-one.

Tibolone (1) (1 g) when reduced with NaBH₄ in dichloromethane yielded reduced products i.e. 3β-hydroxytibolone (2) and 3α-hydroxytibolone (3) in order to check the effect on hydroxylation at various positions in presence of C-3 hydroxyl group (α and β isomers) was investigated. The presence of hydroxyl group at C-3 pronounced the hydroxylation at C-6 as in case of Compounds 17 and 19 and produced better yield of these metabolites as compared to tibolone (1) hydroxyl metabolites. The ¹H NMR spectra of both the isomers 2 and 3 showed OH-bearing geminal methine protons appeared at δ 3.8 (m, W_(1/2)˜21.5 Hz) and 4.04 (m, W_(1/2)˜10.7 Hz), which indicated the reduction of the C-3 ketonic group. The orientation of C-3 methine proton in the both isomers 2 and 3 was deduced from the multiplicity of C-3 proton signals resonated at δ 3.8 (m, W_(1/2)˜21.5 Hz, H-3α) and 4.04 (m, W_(1/2)˜10.7 Hz, H-3β), respectively.

Incubation of 3β-hydroxytibolone (2) (300 mg) with Cunninghamella elegans for twelve days yielded one hydroxyl-bearing Compound 17. Compound 17 showed the M⁺ at m/z 330.2346 corresponding to the formula C₂₁H₃₀O₃ by HREIMS. The ¹H NMR spectrum showed a resonance for OH-beared methine proton at δ 3.50 (d, J=3.5 Hz), while corresponding carbon resonated at δ 74.2 in ¹³C NMR spectrum. The position of the newly introduced hydroxyl group at C-6 was deduced through HMBC interactions. The α (equatorial) orientation of C-6 proton, geminal to the hydroxyl group, was deduced on the basis of NOESY correlations between H-6 (δ 3.50) and C-19 methyl protons (δ 0.73). The structure of Compound 17 was identified as 7α-methyl-17α-ethynl-3β,6β,17β-trihydroxy-19-norandrost-5(10)-en-3-one.

Fermentation of 3α-hydroxytibolone (3) (400 mg) with Cunninghamella elegans for twelve days yielded three polar Compounds 18-20. Compound 18 showed the M⁺ at m/z 314.2541 corresponding to the formula C₂₁H₃₀O₂ by HREIMS, while the ¹H and ¹³C NMR spectra showed appearance of olefinic proton at 5.38 (d, J=4.5 Hz) with corresponding carbon resonated at δ 132.0 as compared to substrate 3 and was assigned to C-6 methine carbon. The β(axial) orientation of C-10 proton was deduced on the basis of NOESY correlations. From these data, the new compound 18 was identified as 7α-methyl-17α-ethynl-3α,17β-dihydroxy-19-norandrost-5-en-3-one.

The HREI-MS of Compound 19 showed the M⁺ at m/z 330.2356 corresponding to the formula C₂₁H₃₀O₃ by HREIMS. The ¹H NMR spectrum of 19 showed one additional OH-bearing methine proton signal at δ 4.13 (br. s), while ¹³C NMR spectrum showed the resonance of corresponding methine carbon at δ74.0 (C-6). The ¹H-¹H COSY 45° spectrum also showed an allylic coupling between H-4 and H-6 (δ 4.13). The α (equatorial) orientation of C-6 proton, geminal to hydroxyl group, was deduced on the basis NOESY correlations between H-6 and Me-19 protons (δ 0.87). The structure of Compound 19 was identified as 7α-methyl-17α-ethynl-3α,6β,17β-trihydroxy-19-norandrost-4-en-3-one.

Compound 20 showed the M⁺ at m/z 330.2256 in HREIMS spectrum in agreement with the formula C₂₁H₃₀O₃. The ¹H and ¹³C NMR spectrum of 20 showed an OH-bearing methine group appeared at δ_(H) 3.80 (ddd, J=13.5, 9.5, 3.3 Hz) and δ_(C) 66.3 and was established for C-11 through homonuclear COSY correlations between H-11 (δ 3.80), H-18 (δ 1.04) and H₂-12 (δ 2.1, 1.64), while HMBC cross-peak correlations revealed the connectivities between H-11, H-18 (δ 1.04) and C-11 (δ 66.3). The β(axial) orientation of C-11 proton, was geminal to hydroxyl group, was deduced on the basis of larger magnitude of coupling constant of H-11 signal at δ_(H) 3.82 (ddd, J=13.5, 9.5, 3.3 Hz) and NOESY correlations between H-11 and Me-19 protons (δ 0.88). The structure of Compound 20 was proposed as 7α-methyl-17α-ethynl-3α,11α,17β-trihydroxy-19-norandrost-4-en-3-one.

Some hydroxy metabolites of tibolone (1) showed significant inhibitory activity against enzyme tyrosinas, mainly hydroxylation at C-6, C-11 and C-15 in Compounds 4, 9 and 10 showed pronounced inhibitory activity against enzyme tyrosinas, while α, β unsaturated system (C-4/C-5) in Compound 6 also showed good activity. These metabolites also showed significant inhibitory activity against enzyme α-Glucosidase enzyme. The hydroxyl group at C-6 position (β isomer in Compound 4) showed potent α-Glucosidase inhibitory activity, while other metabolites containing α, β unsaturated system (C-4/C-5) as in Compound 6 and α, β unsaturated system bearing hydroxyl group at C-6 (α and β isomers), C-10β and C-15β showed pronounced inhibitory activities against enzyme α-Glucosidase enzyme as in Compounds 8, 12 and 13. Other α, β unsaturated systems (C-1/C-2, C-4/C-5) in Compound 7 and hydroxyl bearing moiety in Compound 15 also showed significant activity against enzyme α-Glucosidase enzyme. It was concluded that hydroxylation at C-6 position and α, β unsaturated system pronounced inhibitory activity against α-Glucosidase enzyme.

The fermentation of tibolone (1) by fungus yielded thirteen Compounds 4-16. While incubation of hydroxytibolones (2 and 3) with Cunninghamella elegans is yielded four polar Compounds 17-20. The main hydroxylations occurred in rings B and D, especially at C-6 and C-15(β) positions. The Compounds 4, 5, 9, 10, 12, 13, 14, 15, and 16 were identified as the main metabolites obtained in these fermentations. Compounds 4, 12, 13, and 15 containing an OH-group at C-6 showed pronounced inhibitory activity against α-glucosidase enzyme (Table 2).

TABLE 2 α-Glucosidase inhibitory activities of tibolone (1) and its analogs 4-16, as compared with the reference inhibitors Compound IC₅₀ ± S.E.M.^(a) (in μM)  1 NA^(b)  4 225.0 ± 0.00  5 NA^(b)  6 343.0 ± 0.00  7 227.0 ± 0.02  8 877.0 ± 0.03  9 NA^(b) 10 NA^(b) 11 NA^(b) 12 653.0 ± 0.02 13 <70.0 14 <70.0 15 340.0 ± 0.02 16 NA^(b) Deoxynojirimycin^(c) 425.6 ± 8.14 Acarbose^(c) 780.0 ± 0.28 * Notes: ^(a)S.E.M. is the standard error of the mean; ^(b)are the inactive compounds and ^(c)are the standard inhibitors of the enzyme α-Glucosidase. Compounds 17-20 were not tested due to insufficient quantities.

EXPERIMENTAL DETAILS General

Melting points were determined on a Yanaco MP-S3 apparatus. UV spectra were measured on a Shimadzu UV 240 spectrophotometer. IR spectra were recorded on a JASCO A-302 spectrophotometer in CHCl₃. ¹H- and ¹³C-NMR spectra were recorded on a Bruker Avance AM-400 spectrometer with tetramethylsilane (TMS) as an internal standard. 2D NMR spectra were recorded on a Bruker Avance AMX 500 NMR spectrometer. Optical rotations were measured on JASCO DIP-360 digital polarimeter by using 10 cm cell tube. Mass spectra (EI and HREI-MS) were measured in an electron impact mode on Varian MAT 12 or MAT 312 spectrometers and ions are given in m/z (%). TLC was performed on a pre-coated silica gel card (E. Merck), spots were viewed with ultraviolet light at 254 nm for flouresence quenching spots and at 366 nm for fluorescent spot and stained by spraying with a solution of ceric sulphate in 10% H₂SO₄. For column chromatography, silica gel (E. Merck, 230-400 mesh). Tibolone (1) was extracted from Livial-Organon using dichloromethane.

Fungi and Culture Conditions

Microbial cultures of the Fusarium lini (NRRL 68751), Rhizopus stolonifer (TSY 0471), Cunninghamella elegans (TSY 0865) and Gibberella fujikuroi were grown on Sabouraud-4% glucose-agar (Merck) at 25° C. and stored at 4° C. Rhizopus stolonifer (TSY 0471) medium was prepared by adding glucose (100 g) peptone (25 g), KH₂PO₄ (25 g) and yeast extract (15 g) into distilled water (4 L) and pH was maintained at 5.6. Fusarium lini (NRRL 68751) and Cunninghamella elegans (TSY 0865) media were prepared by mixing the following ingredients into distilled H₂O (3.0 L) in each case: glucose (30.0 g), glycerol (30.0 g), peptone (15.0 g), yeast extract (15.0 g), KH₂PO₄ (15.0 g) and NaCl (15.0 g). Gibberella fujikuroi medium was prepared by adding the following ingredients into distilled H₂O (3.0 L): glucose (80.0 g), KH₂PO₄ (5.0 g), MgSO₄.2H₂O (1.0 g), NH₄NO₃ (0.5 g) and Gibberella trace element solution (2 mL). The Gibberella trace element solution was prepared by mixing Co(NO₃)₂.6H₂O (0.01 g), FeSO₄.7H₂O (0.1 g), CuSO₄.5H₂O (0.1 g), ZnSO₄.7H₂O (0.161 g), MnSO₄.4H₂O (0.01 g) and NH₄ molybdate (0.01 g) into distilled water (100 mL).

General Fermentation and Extraction Conditions

The fungal media were transferred into 250 mL conical flasks (100 mL each) and autoclaved at 121° C. Seed flasks were prepared from three-day old slant and fermentation was allowed for two days on a shaker at 25° C. The remaining flasks were inoculated from seed flasks. After two days, tibolone (1) was dissolved in acetone and transferred in each flask (15 mg/0.5 mL) and the flasks were placed on a rotary shaker (128 rpm) at 22° C. for fermentation period. The time course study was carried out after two days and the transformation was analyzed on TLC. The culture media were filtrated and extracted with CH₂Cl₂. The extract was dried over anhydrous Na₂SO₄, evaporated under reduced pressure and the brown gummy crude was analyzed by thin layer chromatography.

Fermentation of Tibolone (1) with Rhizopus Stolonifer (TSY-0471)

Compound 1 (500 mg), dissolved in 15 mL acetone and distributed among 40 flasks and allowed them for fermentation process. All the media were filtered after 3 days and extracted with dichloromethane and evaporated under reduced pressure to finally yield brown thick crude (0.90 mg), and the transformed metabolites were isolated by using column chromatography. Compound 4 (20 mg) was eluted with petroleum ether and EtOAc (60:40), compound 5 (17 mg) with petroleum ether-EtOAc (58:42) and compound 6 (40 mg) with petroleum ether-EtOAc (55:45).

6β-Hydroxytibolone (4)

White amorphous solid (8.2 mg); mp 186-188° C.; [α]²⁵ _(D)−17 (c 0.35, CHCl₃); UV (MeOH) λ_(max) (log ε) 204 (3.7) nm; IR (CHCl₃) ν_(max) 3381, 2150, 1705, 1668, 1043 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃, Tables 1 and 3; EIMS m/z (rel. int. %) 328 (M⁺, 6), 309 (5), 241 (16), 226 (9), 169 (14), 149 (23), 138 (28), 121 (28), 109 (23), 107 (100), 97 (20), 93 (21), 81 (26), 71 (22), 69 (41), 55 (64); HREIMS m/z 328.2171 (calculated for C₂₁H₂₈O₃, 328.2143).

15β-Hydroxytibolone (5)

White solid (7.6 mg); mp 202-205° C.; [α]²⁵ _(D)+16 (c 0.31, CHCl₃); UV (MeOH)λ_(max) (log ε) 203.4 (3.4) nm; IR (CHCl₃) ν_(max) 3383, 2162, 1708, 1663, 1050 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃, Tables 1 and 3; EIMS m/z (rel. int. %) 328 (M⁺, 3), 312 (100), 245 (27), 229 (36), 203 (17), 189 (14), 187 (17), 174 (24), 161 (28), 149 (26), 135 (24), 121 (25), 96 (38), 81 (23), 69 (21), 67 (24), 55 (59); HREIMS m/z 328.2070 (calculated for C₂₁H₂₈O₃, 328.2038).

Δ⁴-Tibolone (6)

Crystalline solid (20.4 mg); mp 206-2080° C.; [α]²⁵ _(D)−145 (c 0.21, CHCl₃); UV (MeOH) λ_(max) (log ε) 235 (3.2) nm; IR(CHCl₃) ν_(max) 3402, 2150, 1687, 1667, 1017 cm⁻¹; ¹H NMR data in CDCl₃, Tables 1; ¹³C NMR (100 MHz, CDCl₃) δ 198.1 (C-3), 126.4 (C-4), 161.4 (C-5), 38.2 (C-10), 79.1 (C-17), 87.5 (C-20), 74.3 (C-21); EIMS m/z (rel. int. %) 312 (M⁺, 34,), 245 (53), 229 (33), 187 (17), 173 (18), 161 (20), 147 (28), 135 (56), 121 (23), 109 (32), 107 (43), 105 (39), 95 (24), 91 (62), 81 (34), 79 (59), 67 (58), 55 (100); HREIMS m/z 312.2023 (calculated for C₂₁H₂₈O₂, 312.2089).

Fermentation of Tibolone (1) by Fusarium Lini (NRRL 68751) and Cunninghamella Elegans (TSY 0865).

Compound 1 (600 mg), dissolved in 18 mL acetone and distributed among 50 flasks, was kept for fermentation. Fermentation was continued for 6 days and then filtrates were extracted with dichloromethane and evaporated under reduced pressure to afford brown thick crude (1.02 gm). Column chromatography technique was used for the separation of Compounds 7-10 from Cunninghamella elegans crude while Fusarium lini yielded one major Compound 6. Compound 7 (6.2 mg) was eluted with petroleum ether-EtOAc (42:58), compound 8 (15.5 mg) with petroleum ether-EtOAc (38:62), 9 (5.2 mg) with petroleum ether-EtOAc (40:60), whereas compound 10 (10.2 mg) with petroleum ether-EtOAc (30:70).

Δ^(1,4)-Tibolone (7)

Amorphous solid (6.2 mg); mp 196-200° C.; [α]²⁵ _(D)−172 (c 0.32, CHCl₃); UV (MeOH) λ_(max) (log ε) 241.5 (4.1) nm; IR (CHCl₃) ν_(max) 3303, 2154, 1691, 1666, 1044 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃, Tables 1 and 3; EIMS m/z (rel. int. %) 310 (M⁺, 42), 241 (100), 230 (11), 199 (17), 187 (27), 161 (22), 149 (34), 145 (17), 119 (18), 109 (14), 91 (62), 67 (62), 55 (100); HREIMS m/z 310.2004 (calculated for C₂₁H₂₆O₂, 310.2011).

10β-Hydroxy-Δ⁴-tibolone (8)

White powdered solid (15.5 mg); mp 198-201° C.; [α]²⁵ _(D)+12 (c 0.25, CHCl₃); UV (MeOH) λ_(max) (log ε) 239 (2.9) nm; IR (CHCl₃) ν_(max) 3345, 2149, 1698, 1649, 1018 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃, Tables 1 and 3; EIMS m/z (ret. int. %) 328 (M⁺, 13), 310 (14), 229 (20), 187 (25), 171 (26), 161 (25), 149 (48), 136 (32), 124 (55), 109 (44), 107 (43), 91 (55), 83 (28), 67 (47), 57 (50), 55 (100); HREIMS m/z 328.2090 (calculated for C₂₁H₂₈O₃, 328.2123).

11α,15β-Dihydroxytibolone (9)

White powdered solid (5.2 mg); mp 208-210° C.; [α]²⁵ _(D)−81 (c 0.24, CHCl₃); UV (MeOH) λ_(max) (log ε) 203 (3.3) nm; IR (CHCl₃) ν_(max) 3342, 2142, 1718, 1636, 1028 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃, Tables 1 and 3; EIMS m/z (rel. int. %) 344 (M⁺, 13), 310 (14), 229 (20), 187 (25), 171 (26), 161 (25), 149 (48), 136 (32), 124 (55), 109 (44), 107 (43), 91 (55), 83 (28), 67 (47), 57 (50), 55 (100); HREIMS m/z 344.2212 (calculated for C₂₁H₂₈O₄, 344.2234).

11α,15β-Dihydroxy-Δ⁵-tibolone (10)

White powdered solid (7.3 mg); mp 207-211° C.; [α]²⁵ _(D)+27 (c 0.28, CHCl₃); UV (MeOH) λ_(max) (log ε) 202.4 (3.4) nm; IR (CHCl₃) ν_(max) 3312, 2102, 1722, 1652, 1057 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃, Tables 1 and 3; EIMS m/z (rel. int. %) 344 (M⁺, 8), 310(14), 229(20), 187(25), 171 (26), 161 (54), 149(87), 136 (32), 124 (55), 109 (44), 107 (43), 91 (55), 83 (28), 67 (74), 57 (50), 55 (100); HREIMS m/z 344.2341 (calculated for C₂₁H₂₈O₄, 344.2316).

Fermentation of Tibolone (1) by Gibberella Fujikuroi (ATCC 10704)

Compound 1 (850 mg) was dissolved in 20 mL acetone and distributed among 30 flasks for fermentation for 12 days. After fermentation, media was extracted with dichloromethane and evaporated to get a crude extract (1.22 gm). Column chromatography technique was used for the separation of Compounds 11-16 from crude extract. Compound 11 (5.2 mg) was eluted with petroleum ether-EtOAc (54:46), compound 12 (10.2 mg) with petroleum ether-EtOAc (50:50), compound 13 (11.2 mg) with petroleum ether-EtOAc (45:55), compound 14 (8.3 mg) with petroleum ether-EtOAc (38:58), compound 15 (9.5 mg) with petroleum ether-EtOAc (30:70) and compound 16 (10.3 mg) with petroleum ether-EtOAc (35:75).

Δ⁵-Tibolone (11)

White powdered solid (5.6 mg); mp 201-205° C.; [α]²⁵ _(D)−17 (c 0.20, CHCl₃); UV (MeOH) λ_(max) (log ε) 203.6 (3.9) nm; IR (CHCl₃) ν_(max) 3341, 2157, 1728, 1642, 1088 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMS m/z (rel. int. %) 312 (M¹, 42), 242 (100), 227 (63), 187 (27), 161 (22), 149 (34), 124 (26), 91 (21), 67 (23), 55 (100); HREIMS m/z 312.1456 (calculated for C₂₁H₂₈O₂, 312.1513).

6β-Hydroxy-Δ⁴-tibolone (12)

White powdered solid (9.0 mg); mp 189-93° C.; [α]²⁵ _(D)−101 (c 0.41, CHCl₃); UV (MeOH) λ_(max) (log ε) 239.5 (2.1) nm; IR (CHCl₃) ν_(max) 3332, 2128, 1698, 1651, 1063 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃, and 3; EIMS m/z (rel. int. %) 328 (M⁺, 13), 312 (13), 245 (17), 229 (34), 189 (17), 187 (17), 161 (28), 149 (49), 121 (45), 91 (38), 69 (35), 67 (36), 55 (100); HREIMS m/z 328.2176 (calculated for C₂₁H₂₈O₃, 328.2116).

6α-Hydroxy-Δ⁴-tibolone (13)

White powdered solid (7.0 mg); mp 187-190° C.; [α]²⁵ _(D)+66.2 (c 0.35, CHCl₃); UV (MeOH) λ_(max) (log ε) 240.1 (3.7) nm; IR (CHCl₃) ν_(max) 3342, 2106, 1683, 1659, 1061 cm⁻¹; ¹H and ¹³C NMR data in CDCl; EIMS m/z (rel. int. %) 328 (M⁺, 56), 312 (11), 245 (45), 229 (6), 201 (17), 187 (14), 1.71 (20), 161 (34), 149 (57), 135 (24), 121 (46), 91 (34), 81 (34), 67 (4), 55 (59); HREIMS m/z 328.2132 (calculated for C₂₁H₂₈O₃, 328.2205).

15α-Hydroxy-Δ⁴-tibolone (14)

White powdered solid (8.3 mg); mp 203-205° C.; [c]²⁵ _(D)−21.3 (c 0.34, CHCl₃); UV (MeOH) λ_(max) (log ε) 237.6 (2.6) nm; IR (CHCl₃) ν_(max) 3401, 2176, 1691, 1662, 1060 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMS m/z (rel. int. %) 328 (M⁺, 3), 312 (100), 245 (27), 229 (36), 203 (17), 189 (10), 187 (17), 174 (14), 161 (56), 149 (26), 135 (5), 121 (67), 96 (38), 81 (23), 69 (15), 67 (34), 55 (51); HREIMS m/z 328.2212 (calculated for C₂₁H₂₈O₃, 328.2283).

6α-Hydroxy-Δ^(1,4)-tibolone (15)

White powdered solid (5.5 mg); mp 193-196° C.; [α]²⁵ _(D)+44 (c 0.25, CHCl₃); UV (MeOH) λ_(max) (log ε) 244 (3.7) nm; IR (CHCl₃) ν_(max) 3441, 2123, 1680, 1637, 1045 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMS m/z (rel. int. %) 326 (M⁺, 13), 312 (34), 245 (27), 229 (67), 203 (5), 189 (14), 187 (14), 174 (24), 161 (56), 149 (26), 135 (24), 121 (100), 96 (45), 81 (23), 69 (12), 67 (24), 55 (34); HREIMS m/z 326.2231 (calculated for C₂₁H₂₈O₃, 326.2292).

6β-Methoxy-Δ⁴-tibolone (16)

White powdered solid (8.0 mg); mp 206-209° C.; [α]²⁵ _(D)−18 (c 0.42, CHCl₃,); UV (MeOH) λ_(max) (log ε) 242.1 (3.1) nm; IR (CHCl₃) ν_(max) 3323, 2153, 1691, 1661, 1101 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMS m/z (rel. int. %) 343 (M⁺, 6), 312 (17), 245 (27), 229 (32), 203 (17), 189 (14), 187 (17), 174 (25), 161 (28), 149 (26), 135 (24), 121 (25), 91 (100), 81 (23), 69 (21), 67 (23), 55 (45); HREIMS m/z 343.2356 (calculated for C₂₂H₃₀O₃, 343.2338).

Fermentation of 30-Hydroxytibolone (2) by Cunninghamella Elegans (TSY 0865)

Compound 1 (1 g) was dissolved in dry dichloromethane (100 mL) and cooled in an ice bath to 0° C. Sodium borohydride (500 mg) was added to the solution in portions whilst stirring. The mixture was stirred at room temperature for five hours. Acetic acid (50 mL) was destroyed the excess Sodium borohydride and the dichloromethane was removed in vacuo. Water (100 mL) was added to the resulting oily product, and the suspension was extracted with ethyl acetate (1 L). The ethyl acetate extract was washed with sodium hydrogen carbonate solution (300 mL), water and brine. The removal of the solvent on a rotary evaporator afforded crude extract (1.2 g). Column chromatography technique was used for the separation of these hydroxytibolones, compounds 2 and 3.

Compound 2 (300 mg) was eluted with petroleum ether-EtOAc (50:50) and compound 3 (400 mg) was eluted with petroleum ether-EtOAc (48:52). Compound 2 (300 mg), dissolved in 15 mL acetone and distributed among 40 flasks, was kept for fermentation. Fermentation was continued for 12 days and then filtrates were extracted with CH₂Cl₂ and evaporated under reduced pressure to afford brown thick crude (0.81 gm). Column chromatography technique was used for the separation of one hydroxyl Compound 17 from crude extract. Compound 17 (6.2 mg) was eluted with petroleum ether-EtOAc (40:60).

3β,6β-Dihydroxytibolone (17)

White powdered solid (4.2 mg); mp 206-209° C.; [α]²⁵ _(D)+105 (c 0.31, CHCl₃); UV (MeOH)×(log ε) 201 (3.1) nm; IR (CHCl₃) ν_(max) 3353, 2164, 1663, 1078 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMS m/z (rel. int. %) 330 (M⁺, 7), 312 (17), 245 (27), 229 (32), 203 (17), 189 (14), 187 (17), 174 (25), 161 (28), 149 (26), 135 (24), 121 (25), 91 (100), 81 (23), 69 (21), 67 (23), 55 (45); HREIMS m/z 330.2346 (calculated for C₂₁H₃₂O₃, 328.2374).

Fermentation of 3α-Hydroxytibolone (3) by Cunninghamella Elegans (TSY 0865)

Compound 3 (400 mg), dissolved in 18 mL acetone and distributed among 50 flasks, was kept for fermentation. Fermentation was continued for 12 days and then filtrates were extracted with dichloromethane and evaporated under reduced pressure to afford brown thick crude (0.89 gm). Column chromatography technique was used for the separation of hydroxy Compounds 18-20 from crude extract. Compound 18 (11.3 mg) was eluted with petroleum ether-EtOAc (40:60). Compound 19 (5.2 mg) was eluted with petroleum ether-EtOAc (54:46) and compound 20 (10.2 mg) with petroleum ether-EtOAc (50:50).

3α-Hydroxy-Δ⁵-tibolone (18)

White powdered solid (4.6 mg); mp 206-207° C.; [α]²⁵ _(D)+52.2 (c 0.23, CHCl₃); UV (MeOH); λ_(max) (log ε) 204.2 (4.0) nm; IR (CHCl₃) ν_(max) 3313, 2154, 1646, 1038 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMS m/z (rel. int. %) 314 (M^(+,) 8), 312 (17), 245 (27), 229 (32), 203 (17), 189 (14), 187 (17), 174 (25), 161 (28), 149 (21), 135 (24), 121 (23), 91 (11), 81 (23), 69 (21), 67 (21), 55 (45); HREIMS: m/z 314.2541 (calculated for C₂₁H₃₂O₂, 314.2597).

3α,6β-Dihydroxy-Δ⁴-tibolone (19)

White powdered solid (4.2 mg); mp 201-205° C.; [α]²⁵ _(D)−18 (c 0.31, CHCl₃); UV (MeOH); λ_(max) (log ε) 201.2 (2.9) nm; IR(CHCl₃) ν_(max) 3323, 2156, 1665, 1118 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMS m/z (rel. int. %) 330 (M⁺, 6), 312 (17), 245 (27), 229 (32), 203 (17), 189 (14), 187 (14), 174 (25), 161 (21), 149 (26), 135 (22), 121 (25), 91 (100), 81 (23), 69 (21), 67 (23), 55 (45); HREIMS m/z 330.2356 (calculated for C₂₁H₃₂O₃, 330.2338).

3α,11α-Dihydroxy-Δ⁴-tibolone (20)

White powdered solid (5.1 mg); mp 199-201° C.; [α]²⁵ _(D)+61 (c 0.45, CHCl₃); UV (MeOH); λ_(max) (log ε) 202.6 (3.5) nm; IR (CHCl₃) λ_(max) 3323, 2143, 1668, 1105 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMS m/z (rel. int. %) 330 (M⁺, 4), 312 (14), 245 (27), 229 (42), 203 (1.7), 189 (34), 187 (17), 174 (19), 161 (28), 149 (26), 135 (35), 121 (25), 91 (98), 81 (23), 69 (67), 67 (23), 55 (51); HREIMS m/z 330.2256 (calculated for C₂₁H₃₂O₃, 330.2238).

α-Glucosidase Enzyme Inhibition Assay

α-Glucosidase (E.C.3.2.1.20) enzyme inhibition assay has been performed according to the slightly modified method of Matsui et al. α-Glucosidase (E.C.3.2.1.20) from Saccharomyces sp. was purchased from Wako Pure Chemical Industries Ltd. (Wako 076-02841). The inhibition has been measured spectrophotometrically at pH. 6.9 and at 37° C. using 0.7 mM p-nitrophenyl α-D glucopyranoside (PNP-G) as a substrate and 250 m units/mL enzyme in 50 mM sodium phosphate buffer containing 100 mM NaCl. 1-Deoxynojirimycin (0.425 mM) and acarbose (0.78 Mm) were used as positive controls. The increment in absorption at 400 nm due to the hydrolysis of PNP-G by α-glucosidase was monitored continuously with the spectrophotometer (Molecular Devices, USA). The concentrations of the test compounds, which inhibited the hydrolysis of PNP-G by α-glucosidase by 50% (IC₅₀), were determined by monitoring the effect of increasing the concentration of these compounds on the inhibition values. The IC₅₀ values were then calculated using EZ-Fit enzyme kinetics program (Perrella Scientific Inc., Amherst, Mass., U.S.A.).

REFERENCES

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1. A new chemical compound selected from a group consisting of 6β-Hydroxytibolone (C₂₁H₂₈O₃), 15β-Hydroxytibolone (C₂₁H₂₈O₃), Δ^(1,4)-Tibolone (C₂₁H₂₆O₂), 10β-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃), 11α,15β-Dihydroxytibolone (C₂₁H₂₈O₄), 11α,15β-Dihydroxy-Δ⁵-tibolone (C₂₁H₂₈O₄), Δ⁵-Tibolone (C₂₁H₂₈O₂), 6β-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃), 6α-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃), 15α-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃), 6α-Hydroxy-Δ^(1,4)-tibolone (C₂₁H₂₈O₃), 6β-Methoxy-Δ⁴-tibolone (C₂₂H₃₀O₃), 3β,6β-Dihydroxytibolone (C₂₁H₃₂O₃), 3α-Hydroxy-Δ⁵-tibolone (C₂₁H₃₂O₂), 3α,6β-Dihydroxy-Δ⁴-tibolone (C₂₁H₃₂O₃), 3α,11α-Dihydroxy-Δ⁴-tibolone (C₂₁H₃₂O₃), its derivatives, isomers and salts thereof.
 2. A method of inhibiting alpha-glucosidase enzyme by contacting with Δ⁴-Tibolone (C₂₁H₂₈O₂), 6β-Hydroxytibolone (C₂₁H₂₈O₃), Δ^(1,4)-Tibolone (C₂₁H₂₆O₂), 6β-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃), 6α-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃), 15α-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃), 6α-Hydroxy-Δ^(1,4)-tibolone (C₂₁H₂₈O₃).
 3. A method of producing 6β-Hydroxytibolone, 15β-Hydroxytibolone, and Δ⁴-Tibolone by contacting tibolone with Rhizopus stolonifer (TSY 0471) under suitable fermentation conditions.
 4. A method of producing Δ^(1,4)-Tibolone, 10β-Hydroxy-Δ⁴-tibolone, 11α,15β-Dihydroxytibolone, and 11α,15β-Dihydroxy-Δ⁵-tibolone by contacting tibolone with Cunninghamella elegans (TSY 0865) under suitable fermentation conditions.
 5. A method of producing Δ⁴-Tibolone by contacting tibolone with Fusarium lini (NRRL 68751) under suitable fermentation conditions.
 6. A method of producing Δ⁵-Tibolone, 6β-Hydroxy-Δ⁴-tibolone, 6α-Hydroxy-Δ⁴-tibolone, 15α-Hydroxy-Δ⁴-tibolone, 6α-Hydroxy-Δ^(1,4)-tibolone, 6β-Methoxy-Δ⁴-tibolone, by contacting tibolone with Gibberella fujikuroi (ATCC 10704) under suitable fermentation conditions.
 7. A method of producing 3β,6β-Dihydroxytibolone by contacting 3β-Hydroxytibolone Cunninghamella elegans (TSY 0865) under suitable fermentation conditions. A method of producing 3α-Hydroxy-Δ⁵-tibolone, 3α,6β-Dihydroxy-Δ⁴-tibolone, and 3α,11α-Dihydroxy-Δ⁴-tibolone by contacting 3α-Hydroxytibolone with Cunninghamella elegans (TSY 0865) under suitable fermentation conditions.
 8. A method for the treatment of diabetes mellitus wherein a therapeutically effective amount of an alpha-glucosidase inhibitor, selected from a group comprising Δ⁴-Tibolone (C₂₁H₂₈O₂), 6β-Hydroxytibolone (C₂₁H₂₈O₃), Δ^(1,4)-Tibolone (C₂₁H₂₆O₂), 6β-Hydroxy-Δ⁴-tibolone (C₂, H₂₈O₃), 6α-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃), 15α-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃), 6α-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃) and their isomers, derivates and salts is chosen for use in humans and animals, optionally with a pharmaceutically acceptable vehicle. 